貂源綠膿桿菌的分離鑒定及其致病性研究
發(fā)布時(shí)間：2018-09-12 18:05
【摘要】：隨著經(jīng)濟(jì)的發(fā)展,近年來(lái),山東省毛皮動(dòng)物養(yǎng)殖業(yè)發(fā)展迅速,目前已成為我國(guó)毛皮動(dòng)物養(yǎng)殖第一大省,毛皮動(dòng)物飼養(yǎng)量約占全國(guó)的二分之一。但是隨著我省毛皮動(dòng)物養(yǎng)殖業(yè)集約化程度的提高,毛皮動(dòng)物的發(fā)病率持續(xù)上升,疫病防控形勢(shì)日趨嚴(yán)峻,疫病成為制約毛皮動(dòng)物產(chǎn)業(yè)發(fā)展的重要因素,給毛皮動(dòng)物養(yǎng)殖業(yè)造成了很大的經(jīng)濟(jì)損失。水貂出血性肺炎是嚴(yán)重妨礙水貂養(yǎng)殖業(yè)的重要疫病之一,且疫情日趨嚴(yán)重。綠膿桿菌(Pseudomonas aeruginosa,P.aeruginosa)又稱銅綠假單胞菌,是引起水貂出血性肺炎的病原之一,為了有效的防控水貂出血性肺炎,應(yīng)揭示闡明綠膿桿菌對(duì)水貂的致病性。本研究采用常規(guī)細(xì)菌分離鑒定技術(shù),對(duì)43份患出血性肺炎的水貂肺組織進(jìn)行了綠膿桿菌的分離鑒定,同時(shí)選擇綠膿桿菌代表菌株,建立綠膿桿菌致病性研究動(dòng)物模型,來(lái)闡明綠膿桿菌的致病性。1.綠膿桿菌的分離鑒定2013年,在山東省諸城、文登、臨沂等地采集了43份患出血性肺炎的水貂肺組織。通過(guò)常規(guī)細(xì)菌分離技術(shù)對(duì)這43份病料進(jìn)行了細(xì)菌分離培養(yǎng),并采用形態(tài)學(xué)觀察、生化試驗(yàn)、藥敏試驗(yàn)與16SrDNA測(cè)序等方法,對(duì)分離到的細(xì)菌進(jìn)行鑒定。結(jié)果,分離到的5株細(xì)菌,均為綠膿桿菌,分別命名為F1、F5、F6、F8、F10,分離率為11.6%。藥敏試驗(yàn)結(jié)果表明,5株綠膿桿菌均對(duì)青霉素、紅霉素、卡那霉素耐藥。16SrDNA序列同源性分析結(jié)果表明,5株綠膿桿菌與14株參考菌株間的核苷酸同源性為80.9%~100%,5株綠膿桿菌之間的核苷酸同源性為98.9%~99.7%;系統(tǒng)發(fā)育分析顯示,F5、F6、F8、F10與F1均在同一個(gè)大的分支上。2.貂源綠膿桿菌的致病性研究首先以F1株作為代表菌株,對(duì)小鼠進(jìn)行腹腔接種,建立綠膿桿菌對(duì)小鼠的致病性研究動(dòng)物模型。將30只健康小鼠隨機(jī)分成6組,隔離飼養(yǎng),每組5只。第1-5組小鼠分別腹腔接種菌落數(shù)為6.4×107CFU、6.4×106CFU、6.4×105CFU、6.4×104CFU、6.4×103CFU的F1菌液,第6組小鼠作為陰性對(duì)照。攻毒后對(duì)實(shí)驗(yàn)小鼠連續(xù)觀察7天,記錄小鼠的臨床癥狀,對(duì)在實(shí)驗(yàn)期間死亡的小鼠進(jìn)行剖檢,采集心、肝、脾、肺、腎進(jìn)行細(xì)菌的分布檢測(cè)及病理組織學(xué)檢查。結(jié)果,第1、2組小鼠在接種后12h-18h內(nèi)全部死亡,小鼠的心、肝、脾、肺、腎均有不同程度地出血,尤以肺部病變更為嚴(yán)重;病理組織學(xué)檢查可見小鼠心肌出血、肝細(xì)胞壞死、脾臟有大量炎性細(xì)胞、肺臟出血、肺泡壁增厚、腎間質(zhì)有紅細(xì)胞;在上述器官中均分離到了綠膿桿菌,在小鼠肺部含量最高,脾臟中含量最低;測(cè)得F1對(duì)小鼠的LD50為1.6×106CFU/mL。小鼠致病性試驗(yàn)結(jié)果表明綠膿桿菌對(duì)小鼠具有較強(qiáng)的致病力。以F1株作為代表菌株,對(duì)水貂進(jìn)行鼻腔接種,建立綠膿桿菌對(duì)水貂的致病性研究動(dòng)物模型。將20只健康3月齡水貂分成5組,隔離飼養(yǎng),每組4只。第1-4組分別鼻腔接種菌落數(shù)為1.6×108CFU、1.6×106CFU、1.6×104CFU、1.6×102CFU的F1菌液,第5組水貂為陰性對(duì)照,攻毒后連續(xù)觀察14天,對(duì)實(shí)驗(yàn)期間死亡的水貂和實(shí)驗(yàn)結(jié)束時(shí)撲殺的所有水貂進(jìn)行剖檢,采集其心、肝、脾、肺、腎進(jìn)行病理組織學(xué)檢查及細(xì)菌分離。結(jié)果,第1組水貂在人工感染后20h-44h內(nèi)全部死亡,剖檢可見死亡水貂的肺部嚴(yán)重出血、脾臟腫大,在其心、肝、脾、肺、腎中均分離到了綠膿桿菌。病理組織學(xué)檢查可見心肌纖維出血、肝細(xì)胞變性、脾臟出血壞死、肺出血、間質(zhì)增厚。測(cè)得F1對(duì)水貂的LD50為3.2×107CFU/mL;其他人工感染組在接種后的第1-3d表現(xiàn)精神不振、食欲下降等臨床癥狀,但自攻毒后第4d恢復(fù)正常,直至實(shí)驗(yàn)結(jié)束,病理組織學(xué)觀察未見異常,也均未從相關(guān)臟器中分離到綠膿桿菌。實(shí)驗(yàn)結(jié)果表明,綠膿桿菌能夠感染水貂并引起發(fā)病,但致病性不強(qiáng),應(yīng)該還有其他引起水貂出血性肺炎的病因。本研究闡明了綠膿桿菌對(duì)水貂的致病性,為水貂出血性肺炎的防治提供了實(shí)驗(yàn)數(shù)據(jù),奠定了理論基礎(chǔ)。
[Abstract]:With the development of economy, the fur animal breeding industry in Shandong Province has developed rapidly in recent years, and now it has become the largest fur animal breeding Province in China. The amount of fur animal breeding accounts for about half of the whole country. The epidemic is becoming more and more serious. Pseudomonas aeruginosa (P. aeruginosa), also known as Pseudomonas aeruginosa, is one of the important epidemic diseases that seriously hinder mink breeding. In order to prevent and control mink haemorrhagic pneumonia effectively, the pathogenicity of Pseudomonas aeruginosa should be clarified. In this study, 43 mink lung tissues with haemorrhagic pneumonia were isolated and identified by routine bacterial isolation and identification techniques, and the representative strains of Pseudomonas aeruginosa were selected. Pseudomonas aeruginosa pathogenicity animal model was established to clarify the pathogenicity of Pseudomonas aeruginosa. 1. Isolation and identification of Pseudomonas aeruginosa in 2013, 43 mink lung tissues suffering from hemorrhagic pneumonia were collected in Zhucheng, Wendeng, Linyi, Shandong Province. Bacterial isolation and culture of these 43 samples were carried out by routine bacterial isolation technology, and morphological observation was adopted. Results: Five strains of Pseudomonas aeruginosa were isolated, named F1, F5, F6, F8, F10, and the isolation rate was 11.6%. The results of drug susceptibility test showed that five strains of Pseudomonas aeruginosa were resistant to penicillin, erythromycin and kanamycin. The results showed that the nucleotide homology between 5 Pseudomonas aeruginosa strains and 14 reference strains was 80.9%-100%, and the nucleotide homology between 5 Pseudomonas aeruginosa strains was 98.9%-99.7%. Phylogenetic analysis showed that F5, F6, F8, F10 and F1 were all in the same large branch. Thirty healthy mice were randomly divided into 6 groups and fed in isolation with 5 mice in each group. The number of colonies inoculated into the abdominal cavity of mice in groups 1-5 was 6.4 x 107 CFU, 6.4 x 106 CFU, 6.4 x 105 CFU, 6.4 x 104 CFU, 6.4 x 103 CFU. The F1 bacterial solution of mice in group 6 was used as negative control. The clinical symptoms of the mice were observed continuously for 7 days. The heart, liver, spleen, lungs and kidneys of the dead mice were dissected and the distribution of bacteria and pathological histology were examined. The pathological examination showed that myocardial hemorrhage, hepatocyte necrosis, a large number of inflammatory cells in the spleen, pulmonary hemorrhage, alveolar wall thickening, renal interstitial red blood cells; Pseudomonas aeruginosa was isolated from the above organs, the highest content in the lungs of mice, the lowest content in the spleen; the LD50 of F1 in mice was 1.6 *10. The pathogenicity of Pseudomonas aeruginosa in 6 CFU/mL. mice showed that Pseudomonas aeruginosa had strong pathogenicity to mice. The pathogenicity of Pseudomonas aeruginosa to minks was studied by inoculating F1 strain into nasal cavity. Twenty healthy minks aged 3 months were divided into 5 groups and fed in isolation with 4 in each group. The F1 bacterium solution of 1.6 *108 CFU, 1.6 *106 CFU, 1.6 *104 CFU, 1.6 *102 CFU was negative control. The minks in the fifth group were observed continuously for 14 days after poisoning. The minks died during the experiment and all minks killed at the end of the experiment were dissected and their hearts, livers, spleens, lungs and kidneys were collected for pathological examination and bacterial isolation. Pseudomonas aeruginosa was isolated from the heart, liver, spleen, lung and kidney of dead minks. Myocardial fibrous hemorrhage, hepatocyte degeneration, splenic hemorrhage and necrosis, pulmonary hemorrhage and interstitial thickening were observed by histopathology. The LD50 of F1 was 3.2 *107 CFU/mL. Pseudomonas aeruginosa could infect mink and cause disease, but it was pathogenic. This study clarifies the pathogenicity of Pseudomonas aeruginosa to minks and provides experimental data for the prevention and treatment of mink hemorrhagic pneumonia.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.92
本文編號(hào)：2239818
[Abstract]:With the development of economy, the fur animal breeding industry in Shandong Province has developed rapidly in recent years, and now it has become the largest fur animal breeding Province in China. The amount of fur animal breeding accounts for about half of the whole country. The epidemic is becoming more and more serious. Pseudomonas aeruginosa (P. aeruginosa), also known as Pseudomonas aeruginosa, is one of the important epidemic diseases that seriously hinder mink breeding. In order to prevent and control mink haemorrhagic pneumonia effectively, the pathogenicity of Pseudomonas aeruginosa should be clarified. In this study, 43 mink lung tissues with haemorrhagic pneumonia were isolated and identified by routine bacterial isolation and identification techniques, and the representative strains of Pseudomonas aeruginosa were selected. Pseudomonas aeruginosa pathogenicity animal model was established to clarify the pathogenicity of Pseudomonas aeruginosa. 1. Isolation and identification of Pseudomonas aeruginosa in 2013, 43 mink lung tissues suffering from hemorrhagic pneumonia were collected in Zhucheng, Wendeng, Linyi, Shandong Province. Bacterial isolation and culture of these 43 samples were carried out by routine bacterial isolation technology, and morphological observation was adopted. Results: Five strains of Pseudomonas aeruginosa were isolated, named F1, F5, F6, F8, F10, and the isolation rate was 11.6%. The results of drug susceptibility test showed that five strains of Pseudomonas aeruginosa were resistant to penicillin, erythromycin and kanamycin. The results showed that the nucleotide homology between 5 Pseudomonas aeruginosa strains and 14 reference strains was 80.9%-100%, and the nucleotide homology between 5 Pseudomonas aeruginosa strains was 98.9%-99.7%. Phylogenetic analysis showed that F5, F6, F8, F10 and F1 were all in the same large branch. Thirty healthy mice were randomly divided into 6 groups and fed in isolation with 5 mice in each group. The number of colonies inoculated into the abdominal cavity of mice in groups 1-5 was 6.4 x 107 CFU, 6.4 x 106 CFU, 6.4 x 105 CFU, 6.4 x 104 CFU, 6.4 x 103 CFU. The F1 bacterial solution of mice in group 6 was used as negative control. The clinical symptoms of the mice were observed continuously for 7 days. The heart, liver, spleen, lungs and kidneys of the dead mice were dissected and the distribution of bacteria and pathological histology were examined. The pathological examination showed that myocardial hemorrhage, hepatocyte necrosis, a large number of inflammatory cells in the spleen, pulmonary hemorrhage, alveolar wall thickening, renal interstitial red blood cells; Pseudomonas aeruginosa was isolated from the above organs, the highest content in the lungs of mice, the lowest content in the spleen; the LD50 of F1 in mice was 1.6 *10. The pathogenicity of Pseudomonas aeruginosa in 6 CFU/mL. mice showed that Pseudomonas aeruginosa had strong pathogenicity to mice. The pathogenicity of Pseudomonas aeruginosa to minks was studied by inoculating F1 strain into nasal cavity. Twenty healthy minks aged 3 months were divided into 5 groups and fed in isolation with 4 in each group. The F1 bacterium solution of 1.6 *108 CFU, 1.6 *106 CFU, 1.6 *104 CFU, 1.6 *102 CFU was negative control. The minks in the fifth group were observed continuously for 14 days after poisoning. The minks died during the experiment and all minks killed at the end of the experiment were dissected and their hearts, livers, spleens, lungs and kidneys were collected for pathological examination and bacterial isolation. Pseudomonas aeruginosa was isolated from the heart, liver, spleen, lung and kidney of dead minks. Myocardial fibrous hemorrhage, hepatocyte degeneration, splenic hemorrhage and necrosis, pulmonary hemorrhage and interstitial thickening were observed by histopathology. The LD50 of F1 was 3.2 *107 CFU/mL. Pseudomonas aeruginosa could infect mink and cause disease, but it was pathogenic. This study clarifies the pathogenicity of Pseudomonas aeruginosa to minks and provides experimental data for the prevention and treatment of mink hemorrhagic pneumonia.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.92
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